a. Field of the Invention
This invention relates to an improved process for the preparation of 5,6,7,8-tetrahydrofolic acid (hereinafter referred to briefly as tetrahydrofolic acid).
b. Description of the Prior Art
Tetrahydrofolic acid is a coenzyme represented by the structural formula (I) given below. This is a beneficial compound playing an important role in biosynthetic reactions. In enzymic reactions, for example, the N.sup.5,N.sup.10 -formyl derivatives in which tetrahydrofolic acid is combined with formic acid serve as formyl donors, the N.sup.5,N.sup.10 -methylene derivative in which tetrahydrofolic acid is combined with formaldehyde serves as a hydroxymethyl donor for converting glycine to serine, and the N.sup.5 -methyl derivative which is the reduced form of the methylene derivative serves to convert homocysteine to methionine. ##STR1## Conventionally, a number of processes for the preparation of tetrahydrofolic acid are known. One of them comprises dissolving folic acid in an aqueous solution of sodium hydroxide, reducing it with sodium hydrosulfite to form dihydrofolic acid, and further reducing it with sodium borohydride or the like to form tetrahydrofolic acid [Helv. Chim. Acta, 1980, 63(8), 2554] and another comprises reducing folic acid in acetic acid by means of NaCNBH.sub.3 [Anal. Biochem., 1980, 103(2), 255]. However, these processes are not suitable for the purpose of producing tetrahydrofolic acid on an industrial scale because they have the disadvantages of requiring an expensive reducing agent in large amounts and involving troublesome procedures.
On the other hand, processes for the preparation of tetrahydrofolic acid by hydrogenating folic acid in the presence of a noble metal catalyst are also known. For example, U.S. Pat. Nos. 2,717,250 (1955) and 2,790,802 (1957) disclose a process comprising suspending folic acid in glacial acetic acid and reducing it in the presence of a platinum oxide catalyst. In this process, however, the amount of catalyst used is as much as 10 to 100% by weight based on the folic acid and a large amount of glacial acetic acid is used as the reaction solvent. Moreover, it requires a troublesome procedure for isolating the product. Furthermore, the yield of the product is as low as about 48%. Accordingly, this process cannot be regarded as economically beneficial.
In addition, an instance is found in which folic acid was dissolved in a large amount, i.e. about 19 moles per mole of the folic acid, of an aqueous sodium hydroxide solution (specifically, 36 mg of folic acid was dissolved in 15 cc of 0.1 N NaOH) and its hydrogenation was carried out under such strongly alkaline conditions in the presence of a platinum oxide catalyst used in an amount of as much as 70% by weight based on the folic acid [J. Am. Chem. Soc., 69, 250 (1947)]. In this instance, however, the hydrogenation stopped at the stage of dihydrofolic acid in spite of the large amount of catalyst used, so that only dihydrofolic acid was obtained in low yield and no tetrahydrofolic acid was produced.
Tetrahydrofolic acid is an unstable compound. With the lapse of time, tetrahydrofolic acid is decomposed by the action of oxygen, heat, light and the like to form dihydrofolic acid, folic acid, p-aminobenzoic acid and other compounds. Thus, where tetrahydrofolic acid has undergone a marked degree of deterioration, it sometimes inhibits enzymic reactions. Accordingly, the procedure for isolating tetrahydrofolic acid from the reaction solution obtained by catalytic hydrogenation of folic acid is carried out in an atmosphere of an inert gas (for example, in a box purged with nitrogen gas to replace the air present therein) in order to prevent the tetrahydrofolic acid from being deteriorated by oxidation.
Where the tetrahydrofolic acid thus obtained is in powder form, it is sealed in ampules filled with an inert gas such as argon gas and the like. Where it is in the form of a solution, it is dissolved in an aqueous solution containing 1 mole/liter of mercaptoethanol so as to give a typical concentration of the order of 0.5 g/3 ml, and sealed in ampules filled with an inert gas in the same manner as for powder products. These ampules are solid in a refrigerated state.
As described above, the conventionally known processes for the preparation of tetrahydrofolic acid have not been satisfactory for the purpose of producing it on an industrial scale because hydrogenolysis and the like may cause the formation of by-products during hydrogenation and the reaction mixture may contain residual unreacted folic acid or dihydro compound, resulting in a low yield of the desired product.
Moreover, the conventional methods for stabilizing and storing tetrahydrofolic acid have been effective to a certain degree, but not entirely satisfactory. Specifically, even if a stabilizer such as mercaptoethanol or the like is contained, the stability of a tetrahydrofolic acid solution depends largely on temperature. That is, it undergoes deterioration even at relatively low temperatures around 0.degree. C. and especially remarkable deterioration in quality at room temperature and its vicinities, indicating that the stabilizer does not function properly. Though the storage stability of a powder is better than that of a solution, its deterioration in quality with time has also been unavoidable because it is difficult to substitute an inert gas for the air present in the container in cases where the unused portion of the tetrahydrofolic acid taken out of an ampule is stored again or tetrahydrofolic acid prepared by the user is stored in a container for purposes of captive consumption. Accordingly, it would be desirable to improve the storage stability of tetrahydrofolic acid.